Soluble and insoluble α-glucan synthesis in yeast by enzyme suites derived exclusively from maize endosperm.

Molecular mechanisms that distinguish the synthesis of semi-crystalline α-glucan polymers found in plant starch granules from the synthesis of water-soluble polymers by nonplant species are not well understood. To address this, starch biosynthetic enzymes from maize (Zea mays L.) endosperm were isolated in a reconstituted environment using yeast (Saccharomyces cerevisiae) as a test bed. Ninety strains were constructed containing unique combinations of 11 synthetic transcription units specifying maize starch synthase (SS), starch phosphorylase (PHO), starch branching enzyme (SBE), or isoamylase-type starch debranching enzyme (ISA). Soluble and insoluble branched α-glucans accumulated in varying proportions depending on the enzyme suite, with ISA function stimulating distribution into the insoluble form. Among the SS isoforms, SSIIa, SSIII, and SSIV individually supported the accumulation of glucan polymer. Neither SSI nor SSV alone produced polymers; however, synergistic effects demonstrated that both isoforms can stimulate α-glucan accumulation. PHO did not support α-glucan production by itself, but it had either positive or negative effects on polymer content depending on which SS or a combination thereof was present. The complete suite of maize enzymes generated insoluble particles resembling native starch granules in size, shape, and crystallinity. Ultrastructural analysis revealed a hierarchical assembly starting with subparticles of approximately 50 nm diameter that coalesce into discrete structures of approximately 200 nm diameter. These are assembled into semi-crystalline α-glucan superstructures up to 4 µm in length filling most of the yeast cytosol. ISA was not essential for the formation of such particles, but their abundance was increased dramatically by ISA presence.

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm.

Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.

Effects of long-term exposure to elevated temperature on Zea mays endosperm development during grain fill.

Cereal yields decrease when grain fill proceeds under conditions of prolonged, moderately elevated temperatures. Endosperm-endogenous processes alter both rate and duration of dry weight gain, but underlying mechanisms remain unclear. Heat effects could be mediated by either abnormal, premature cessation of storage compound deposition or accelerated implementation of normal development. This study used controlled environments to isolate temperature as the sole environmental variable during Zea mays kernel-fill, from 12 days after pollination to maturity. Plants subjected to elevated day, elevated night temperatures (38°C day, 28°C night (38/28°C])) or elevated day, normal night (38/17°C), were compared with those from controls grown under normal day and night conditions (28/17°C). Progression of change over time in endosperm tissue was followed to dissect contributions at multiple levels, including transcriptome, metabolome, enzyme activities, product accumulation, and tissue ultrastructure. Integrated analyses indicated that the normal developmental program of endosperm is fully executed under prolonged high-temperature conditions, but at a faster rate. Accelerated development was observed when both day and night temperatures were elevated, but not when daytime temperature alone was increased. Although transcripts for most components of glycolysis and respiration were either upregulated or minimally affected, elevated temperatures decreased abundance of mRNAs related to biosynthesis of starch and storage proteins. Further analysis of 20 central-metabolic enzymes revealed six activities that were reduced under high-temperature conditions, indicating candidate roles in the observed reduction of grain dry weight. Nonetheless, a striking overall resilience of grain filling in the face of elevated temperatures can be attributed to acceleration of normal endosperm development.

Characterizing the Physical Properties and Cell Compatibility of Phytoglycogen Extracted from Different Sweet Corn Varieties.

Owing to its unique structure and properties, the glucose dendrimer phytoglycogen is gaining interest for medical and biotechnology applications. Although many maize variants are available from commercial and academic breeding programs, most applications rely on phytoglycogen extracted from the common maize variant, sugary1. Here we characterized the solubility, hydrodynamic diameter, water-binding properties, protein contaminant concentration, and cytotoxicity of phytoglycogens from different maize sources, A632su1, A619su1, Wesu7, and Ia453su1, harboring various sugary1 mutants. A619su1-SW phytoglycogen was cytotoxic while A632su1-SW phytoglycogen was not. A632su1-Pu phytoglycogen promoted cell growth, whereas extracts from A632su1-NE, A632su1-NC, and A632su1-CM were cytotoxic. Phytoglycogen extracted from Wesu7su1-NE using ethanol precipitation was cytotoxic. Acid-treatment improved Wesu7 phytoglycogen cytocompatibility. Protease-treated Wesu7 extracts promoted cell growth. Phytoglycogen extracted from Ia453su1 21 days after pollination ("Ia435su1 21DAP") was cytotoxic, whereas phytoglycogen extracted at 40 days ("Ia435su1 40DAP") was not. In general, size and solubility had no correlation with cytocompatibility, whereas protein contaminant concentration and water-binding properties did. A632su1-CM had the highest protein contamination among A632 mutants, consistent with its higher cytotoxicity. Likewise, Ia435su1 21DAP phytoglycogen had higher protein contamination than Ia435su1 40DAP. Conversely, protease-treated Wesu7 extracts had lower protein contamination than the other Wesu7 extracts. A632su1-NE, A632su1-NC, and A632su1-CM had similar water-binding properties which differed from those of A632su1-Pu and A632su1-SW. Likewise, water binding differed between Ia435su1 21DAP and Ia435su1 40DAP. Collectively, these data demonstrate that maize phytoglycogen extracts are not uniformly cytocompatible. Rather, maize variant, plant genotype, protein contaminants, and water-binding properties are determinants of phytoglycogen cytotoxicity.

Fundamental differences in starch synthesis in the maize leaf, embryo, ovary and endosperm.

Enzymological and starch analyses of various ADP-glucose pyrophosphorylase (AGPase) null mutants point to fundamental differences in the pathways for starch synthesis in the maize leaf, embryo, ovary and endosperm. Leaf starch is synthesized via the AGPase encoded by the small and large subunits shown previously to be expressed at abundant levels in the leaf, whereas more than one AGPase isoform functions in the embryo and in the ovary. Embryo starch content is also dependent on genes functioning in the leaf and in the endosperm. AGPase encoded by shrunken-2 and brittle-2 synthesizes ~75% of endosperm starch. The gene, agpsemzm, previously shown to encode the small subunit expressed in the embryo, and agpllzm, the leaf large subunit gene, are here shown to encode the endosperm, plastid-localized AGPase. Loss of this enzyme does not reduce endosperm starch. Rather, the data suggest that AGPase-independent starch synthesis accounts for ~25% of endosperm starch. Three maize genes encode the small subunit of the AGPase. Data here show that the triple mutant lacking all three small subunits is lethal in early seed development but can be viable in both male and female gametes. Seed and plant viability is restored by any one of the three small subunit genes, including one previously thought to function only in the cytosol of the endosperm. Data herein also show the functionality of a fourth gene encoding the large subunit of this enzyme. Although adenosine diphosphate glucose pyrophosphorylase is shown here to be essential for maize viability, strong evidence for starch synthesis in the endosperm that is independent of this enzyme is also presented. Starch synthesis is distinct in the maize embryo, ovary, leaf and endosperm, and is coordinated among the various tissues.

Enhancing the heat stability and kinetic parameters of the maize endosperm ADP-glucose pyrophosphorylase using iterative saturation mutagenesis.

Iterative saturation mutagenesis (ISM) has been used to improve the thermostability of maize endosperm ADP-glucose pyrophosphorylase (AGPase), a highly-regulated, rate-limiting and temperature-sensitive enzyme essential for starch biosynthesis. The thermo-sensitivity of heterotetrameric AGPase has been linked to grain loss in cereals and improving this property might therefore have direct impacts on grain yield. Nine amino acids were selected for site-saturation mutagenesis on the basis of elevated B-factors in the crystal structure of the closest available homolog (a small subunit homotetramer of potato AGPase). After each round of mutagenesis, iodine staining and antibody capture activity assays at varying temperatures were used to select the optimum positions and amino acid changes for the next rounds of mutagenesis. After three iterations, the signals from whole-colony iodine staining were saturated and a heat stable AGPase variant was obtained. Kinetic studies of the heat stable mutant showed that it also had an unexpected increased affinity for the activator, 3-PGA. This is particularly valuable as both the temperature stability and allosteric properties of AGPase significantly influence grain yield.

Enhanced heat stability and kinetic parameters of maize endosperm ADPglucose pyrophosphorylase by alteration of phylogenetically identified amino acids.

ADP-glucose pyrophosphorylase (AGPase) controls the rate-limiting step in starch biosynthesis and is regulated at various levels. Cereal endosperm enzymes, in contrast to other plant AGPases, are particularly heat labile and transgenic studies highlight the importance of temperature for cereal yield. Previously, a phylogenetic approach identified Type II and positively selected amino acid positions in the large subunit of maize endosperm AGPase. Glycogen content, kinetic parameters and heat stability were measured in AGPases having mutations in these sites and interesting differences were observed. This study expands on our earlier evolutionary work by determining how all Type II and positively selected sites affect kinetic constants, heat stability and catalytic rates at increased temperatures. Variants with enhanced properties were identified and combined into one gene, designated Sh2-E. Enhanced properties include: heat stability, enhanced activity at 37 °C, activity at 55 °C, reduced Ka and activity in the absence of activator. The resulting enzyme exhibited all improved properties of the various individual changes. Additionally, Sh2-E was expressed with a small subunit variant with enhanced enzyme properties resulting in an enzyme that has exceptional heat stability, a high catalytic rate at increased temperatures and significantly decreased Km values for both substrates in the absence of the activator.

Sweet corn phytoglycogen dendrimers as a lyoprotectant for dry-state protein storage.

Protein biotherapeutics typically require expensive cold-chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold-chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., "cryoprotection" and "lyoprotection"), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake-forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1-16) were extracted via ethanol precipitation. PG size was generally consistent at ~70-100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1-16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, ß-galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate-buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein-PG13 mixtures had a cake-like appearance and all frozen protein-PG13 mixtures had a Tg' of ~ -26°C. The lyoprotection and cake-forming properties of PG13 were density-dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.

Deciphering the kinetic mechanisms controlling selected plant ADP-glucose pyrophosphorylases.

ADP-Glc pyrophosphorylase (AGPase), a rate-limiting enzyme in starch biosynthesis, is controlled by thermostability and allosteric regulation. Previous studies suggested that redox affects turnover number and heat stability of AGPases. Here, we investigated how allostery and redox state affect kinetic mechanisms of the reduced, heat labile and the oxidized, heat stable potato tuber enzymes; the heat labile maize endosperm enzyme and a chimeric maize/potato heat stable enzyme that lacks the cysteine responsible for redox changes. With 3-PGA, all AGPases followed a Theorell-Chance Bi Bi mechanism with ATP binding first and ADP-Glc releasing last. 3-PGA increases the binding affinity for both substrates with little effect on velocity for the maize and MP isoforms. By contrast, 3-PGA increases the velocity and the affinity for G-1-P for the potato enzymes. Redox state does not affect kcat of the two potato isoforms. Without 3-PGA the oxidized potato enzyme exhibits a rapid equilibrium random Bi Bi mechanism with a dead end ternary complex. This fundamental change from rapid, ordered binding with little buildup of intermediates to a mechanism featuring relatively slow, random binding is unique to the oxidized potato tuber enzyme. Finally, ADP-Glc the physiologically relevant product of this enzyme has complex, isoform-specific effects on catalysis.

The potato tuber, maize endosperm and a chimeric maize-potato ADP-glucose pyrophosphorylase exhibit fundamental differences in Pi inhibition.

ADP-glucose pyrophosphorylase (AGPase) is highly regulated by allosteric effectors acting both positively and negatively. Enzymes from various sources differ, however, in the mechanism of allosteric regulation. Here, we determined how the effector, inorganic phosphate (Pi), functions in the presence and absence of saturating amounts of the activator, 3-phosphoglyceric acid (3-PGA). This regulation was examined in the maize endosperm enzyme, the oxidized and reduced forms of the potato tuber enzyme as well as a small subunit chimeric AGPase (MP), which contains both maize endosperm and potato tuber sequences paired with a wild-type maize large subunit. These data, combined with our previous kinetic studies of these enzymes led to a model of Pi inhibition for the various enzymes. The Pi inhibition data suggest that while the maize enzyme contains a single effector site that binds both 3-PGA and Pi, the other enzymes exhibit more complex behavior and most likely have at least two separate interacting binding sites for Pi. The possible physiological implications of the differences in Pi inhibition distinguishing the maize endosperm and potato tuber AGPases are discussed.

Insights into Mycoplasma genitalium metabolism revealed by the structure of MG289, an extracytoplasmic thiamine binding lipoprotein.

Mycoplasma genitalium is one of the smallest organisms capable of self-replication and its sequence is considered a starting point for understanding the minimal genome required for life. MG289, a putative phosphonate substrate binding protein, is considered to be one of these essential genes. The crystal structure of MG289 has been solved at 1.95 Å resolution. The structurally identified thiamine binding region reveals possible mechanisms for ligand promiscuity. MG289 was determined to be an extracytoplasmic thiamine binding lipoprotein. Computational analysis, size exclusion chromatography, and small angle X-ray scattering indicates that MG289 homodimerizes in a concentration-dependant manner. Comparisons to the thiamine pyrophosphate binding homolog Cypl reveal insights into the metabolic differences between mycoplasmal species including identifying possible kinases for cofactor phosphorylation and describing the mechanism of thiamine transport into the cell. These results provide a baseline to build our understanding of the minimal metabolic requirements of a living organism.

Probing allosteric binding sites of the maize endosperm ADP-glucose pyrophosphorylase.

Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a nonnative, low-activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) reported previously by others revealed that several sulfate ions bind to each enzyme. These sites are also believed to interact with allosteric regulators such as inorganic phosphate and 3-phosphoglycerate (3-PGA). Several arginine (Arg) side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine-scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady-state kinetic and regulatory parameters were measured for each mutant. All of the Arg mutants examined--in both the small and large subunits--bound 3-PGA more weakly than the wild type (A(50) increased by 3.5- to 20-fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA. In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the Arg mutants. Phosphate binding was also affected by Arg substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and inorganic phosphate compete directly.

Studies of the kinetic mechanism of maize endosperm ADP-glucose pyrophosphorylase uncovered complex regulatory properties.

ADP-glucose pyrophosphorylase catalyzes the synthesis of ADP-glucose (ADP-Glc) from Glc-1-phosphate (G-1-P) and ATP. Kinetic studies were performed to define the nature of the reaction, both in the presence and absence of allosteric effector molecules. When 3-phosphoglycerate (3-PGA), the putative physiological activator, was present at a saturating level, initial velocity studies were consistent with a Theorell-Chance BiBi mechanism and product inhibition data supported sequential binding of ATP and G-1-P, followed by ordered release of pyrophosphate and ADP-Glc. A sequential mechanism was also followed when 3-PGA was absent, but product inhibition patterns changed dramatically. In the presence of 3-PGA, ADP-Glc is a competitive inhibitor with respect to ATP. In the absence of 3-PGA--with or without 5.0 mm inorganic phosphate--ADP-Glc actually stimulated catalytic activity, acting as a feedback product activator. By contrast, the other product, pyrophosphate, is a potent inhibitor in the absence of 3-PGA. In the presence of subsaturating levels of allosteric effectors, G-1-P serves not only as a substrate but also as an activator. Finally, in the absence of 3-PGA, inorganic phosphate, a classic inhibitor or antiactivator of the enzyme, stimulates enzyme activity at low substrate by lowering the K(M) values for both substrates.

Detection of antibodies directed at M. hyorhinis p37 in the serum of men with newly diagnosed prostate cancer.

BACKGROUND: Recent epidemiologic, genetic, and molecular studies suggest infection and inflammation initiate certain cancers, including cancers of the prostate. Over the past several years, our group has been studying how mycoplasmas could possibly initiate and propagate cancers of the prostate. Specifically, Mycoplasma hyorhinis encoded protein p37 was found to promote invasion of prostate cancer cells and cause changes in growth, morphology and gene expression of these cells to a more aggressive phenotype. Moreover, we found that chronic exposure of benign human prostate cells to M. hyorhinis resulted in significant phenotypic and karyotypic changes that ultimately resulted in the malignant transformation of the benign cells. In this study, we set out to investigate another potential link between mycoplasma and human prostate cancer. METHODS: We report the incidence of men with prostate cancer and benign prostatic hyperplasia (BPH) being seropositive for M. hyorhinis. Antibodies to M. hyorhinis were surveyed by a novel indirect enzyme-linked immunosorbent assay (ELISA) in serum samples collected from men presenting to an outpatient Urology clinic for BPH (N = 105) or prostate cancer (N = 114) from 2006-2009. RESULTS: A seropositive rate of 36% in men with BPH and 52% in men with prostate cancer was reported, thus leading us to speculate a possible connection between M. hyorhinis exposure with prostate cancer. CONCLUSIONS: These results further support a potential exacerbating role for mycoplasma in the development of prostate cancer.

Genetic Perturbation of the Starch Biosynthesis in Maize Endosperm Reveals Sugar-Responsive Gene Networks.

In maize, starch mutants have facilitated characterization of key genes involved in endosperm starch biosynthesis such as large subunit of AGPase Shrunken2 (Sh2) and isoamylase type DBE Sugary1 (Su1). While many starch biosynthesis enzymes have been characterized, the mechanisms of certain genes (including Sugary enhancer1) are yet undefined, and very little is understood about the regulation of starch biosynthesis. As a model, we utilize commercially important sweet corn mutations, sh2 and su1, to genetically perturb starch production in the endosperm. To characterize the transcriptomic response to starch mutations and identify potential regulators of this pathway, differential expression and coexpression network analysis was performed on near-isogenic lines (NILs) (wildtype, sh2, and su1) in six genetic backgrounds. Lines were grown in field conditions and kernels were sampled in consecutive developmental stages (blister stage at 14 days after pollination (DAP), milk stage at 21 DAP, and dent stage at 28 DAP). Kernels were dissected to separate embryo and pericarp from the endosperm tissue and 3' RNA-seq libraries were prepared. Mutation of the Su1 gene led to minimal changes in the endosperm transcriptome. Responses to loss of sh2 function include increased expression of sugar (SWEET) transporters and of genes for ABA signaling. Key regulators of starch biosynthesis and grain filling were identified. Notably, this includes Class II trehalose 6-phosphate synthases, Hexokinase1, and Apetala2 transcription factor-like (AP2/ERF) transcription factors. Additionally, our results provide insight into the mechanism of Sugary enhancer1, suggesting a potential role in regulating GA signaling via GRAS transcription factor Scarecrow-like1.

Structural insights into the extracytoplasmic thiamine-binding lipoprotein p37 of Mycoplasma hyorhinis.

The Mycoplasma hyorhinis protein p37 has been implicated in tumorigenic transformation for more than 20 years. Though there are many speculations as to its function, based solely on sequence homology, the issue has remained unresolved. Presented here is the 1.6-A-resolution refined crystal structure of M. hyorhinis p37, renamed the extracytoplasmic thiamine-binding lipoprotein (Cypl). The structure shows thiamine pyrophosphate (TPP) and two calcium ions are bound to Cypl and give the first insights into possible functions of the Cypl-like family of proteins. Sequence alignments of Cypl-like proteins between several different species of mycoplasma show that the thiamine-binding site is likely conserved and structural alignments reveal the similarity of Cypl to various binding proteins. While the experimentally determined function of Cypl remains unknown, the structure shows that the protein is a TPP-binding protein, opening up many avenues for future mechanistic studies and making Cypl a possible target for combating mycoplasma infections and tumorigenic transformation.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of Mycoplasma genitalium protein MG289.

Mycoplasma genitalium is a human pathogen that is associated with nongonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 A resolution. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 A. The diffraction data after processing had an overall R(merge) of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

Maize defective kernel5 is a bacterial TamB homologue required for chloroplast envelope biogenesis.

Chloroplasts are of prokaryotic origin with a double-membrane envelope separating plastid metabolism from the cytosol. Envelope membrane proteins integrate chloroplasts with the cell, but envelope biogenesis mechanisms remain elusive. We show that maize defective kernel5 (dek5) is critical for envelope biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. The DEK5 protein is homologous to rice SSG4, Arabidopsis thaliana EMB2410/TIC236, and Escherichia coli tamB. TamB functions in bacterial outer membrane biogenesis. DEK5 is localized to the envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Proteomics and antibody-based analyses show dek5 reduces levels of Toc75 and chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable transport of metabolites and proteins.

Structure determination of the cancer-associated Mycoplasma hyorhinis protein Mh-p37.

The crystal structure of the Mycoplasma hyorhinis protein Mh-p37 has been solved and refined to 1.9 A resolution. This is the first de novo structure to be determined using the recently described heavy-atom reagent [Beck et al. (2008), Acta Cryst. D64, 1179-1182] 5-amino-2,4,6-triiodoisophthalic acid (I3C), which contains three I atoms arranged in an equilateral triangle, by SIRAS methods. Data collection was performed in-house at room temperature. SHELXD and SHELXE were used to determine the I-atom positions and phase the native protein and PHENIX AutoBuild software was used to automatically fit the amino-acid sequence to the electron-density map. The structure was refined using SHELX97 to an R(cryst) of 18.6% and an R(free) of 24.0%. Mh-p37 is an alpha/beta protein with two well defined domains which are separated by a deep cleft. An unanticipated ligand bound in the center of the molecule at the base of the cleft has been modeled as thiamine pyrophosphate or vitamin B(1). Retrospective attempts to solve the crystal structure by Patterson search methods using either isomorphous or anomalous differences failed. Additionally, attempts to use proteins with the highest structural homology in the Protein Data Bank to phase the data by molecular replacement were unsuccessful, most likely in hindsight because of their poor structural agreement. Therefore, the I3C reagent offers an alternative, quick and inexpensive method for in-house phasing of de novo structures where other methods may not be successful.

Enhancement of Heat Stability and Kinetic Parameters of the Maize Endosperm ADP-Glucose Pyrophosphorylase by Mutagenesis of Amino Acids in the Small Subunit With High B Factors.

ADP-glucose pyrophosphorylase (AGPase) is an important enzyme in starch synthesis and previous studies showed that the heat lability of this enzyme is a determinant to starch synthesis in the maize endosperm and, in turn, seed yield. Here, amino acids in the AGPase endosperm small subunit with high B-factors were mutagenized and individual changes enhancing heat stability and/or kinetic parameters in an Escherichia coli expression system were chosen. Individual mutations were combined and analyzed. One triple mutant, here termed Bt2-BF, was chosen for further study. Combinations of this heat stable, 3-PGA-independent small subunit variant with large subunits also heat stable yielded complex patterns of heat stability and kinetic and allosteric properties. Interestingly, two of the three changes reside in a protein motif found only in AGPases that exhibit high sensitivity to 3-PGA. While not the 3-PGA binding site, amino acid substitutions in this region significantly alter 3-PGA activation kinetics.